Abstract

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.

A) Thrombus formation was initiated by topical application of FeCl3 on mesenteric arterioles in C57BL/6 male mice, which were injected with fluorescently-labeled platelets and different concentration of Dp-3-g or control buffer. Thrombus formation was compared between groups based on the time to complete vessel occlusion. Values are mean ± SD, n = 6–10 per group. *** P<0.001, as compared to control buffer. B) C57BL/6 mice were injected with 50 µM Dp-3-g or control buffer. Blood flow in the carotid artery following FeCl3-induced injury was monitored until complete vessel occlusion was observed. Values are mean ± SD, n = 14 per group. * P<0.05.

Tail-vein bleeding times were examined in C57BL/6 mice. Either PBS (control) or different concentrations of Dp-3-g were administered via the tail vein 40 min before the bleeding time was determined. Values are mean ± SD, n = 8–10 per group. No significant differences in bleeding times were observed between treated and untreated mice.

Effects of Dp-3-g on human and murine platelet phosphorylation of threonine residues of AMPK.

Platelets activated with collagen in the presence of Dp-3-g were lysed and proteins were separated by SDS–PAGE and immunoblotted to detect phospho-threonine residues. A) Western blot analysis of AMPK phosphorylation in human platelets and the associated percent-change observed when compared with controls. B) Western blot analysis of AMPK phosphorylation in murine platelets and the associated percent-change observed when compared with controls. Values are mean ± SD, n = 3 per group. * P<0.05 and ** P<0.01, as compared to control buffer.